C/EBPβ is an auto‐repressed protein that becomes post‐translationally activated by Ras‐MEK‐ERK signalling. C/EBPβ is required for oncogene‐induced senescence (OIS) of primary fibroblasts, but also displays pro‐oncogenic functions in many tumour cells. Here, we show that C/EBPβ activation by H‐RasV12 is suppressed in immortalized/transformed cells, but not in primary cells, by its 3′ untranslated region (3′UTR). 3′UTR sequences inhibited Ras‐induced cytostatic activity of C/EBPβ, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3′UTR suppressed induction of senescence‐associated C/EBPβ target genes, while promoting expression of genes linked to cancers and TGFβ signalling. An AU‐rich element (ARE) and its cognate RNA‐binding protein, HuR, were required for 3′UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPβ translation controls de‐repression by Ras signalling. Notably, 3′UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras‐induced C/EBPβ activation and OIS to proceed. Our findings reveal a novel mechanism whereby non‐coding mRNA sequences selectively regulate C/EBPβ activity and suppress its anti‐oncogenic functions.

eThe integrated stress response (ISR) controls cellular adaptations to nutrient deprivation, redox imbalances, and endoplasmic reticulum (ER) stress. ISR genes are upregulated in stressed cells, primarily by the bZIP transcription factor ATF4 through its recruitment to cis-regulatory C/EBP:ATF response elements (CAREs) together with a dimeric partner of uncertain identity. Here, we show that C/EBP␥:ATF4 heterodimers, but not C/EBP␤:ATF4 dimers, are the predominant CARE-binding species in stressed cells. C/EBP␥ and ATF4 associate with genomic CAREs in a mutually dependent manner and coregulate many ISR genes. In contrast, the C/EBP family members C/EBP␤ and C/EBP homologous protein (CHOP) were largely dispensable for induction of stress genes. Cebpg ؊/؊ mouse embryonic fibroblasts (MEFs) proliferate poorly and exhibit oxidative stress due to reduced glutathione levels and impaired expression of several glutathione biosynthesis pathway genes. Cebpg ؊/؊ mice (C57BL/6 background) display reduced body size and microphthalmia, similar to ATF4-null animals. In addition, C/EBP␥-deficient newborns die from atelectasis and respiratory failure, which can be mitigated by in utero exposure to the antioxidant N-acetylcysteine. Cebpg ؊/؊ mice on a mixed strain background showed improved viability but, upon aging, developed significantly fewer malignant solid tumors than WT animals. Our findings identify C/EBP␥ as a novel antioxidant regulator and an obligatory ATF4 partner that controls redox homeostasis in normal and cancerous cells.A variety of stresses, such as amino acid limitation, protein misfolding in the endoplasmic reticulum (ER), oxidative stress, hypoxia, and intracellular pathogens, activate gene expression programs collectively known as the integrated stress response (ISR) (1). Stress-induced genes are involved in multiple cellular processes that include nutrient uptake, amino acid synthesis, metabolic changes, antioxidant defenses, and cell survival, leading to cell recovery and alleviation of stress. However, prolonged or irresolvable stress can trigger cell death (2).Protein misfolding and ER stress are associated with several diseases, including diabetes and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Huntington's disease (3). The ISR also plays an important role in cancer as tumor cells frequently experience nutrient deprivation, hypoxia, and oxidative stress and require stress response regulators such as the transcription factor (TF) ATF4 to thrive under adverse conditions (4, 5). Elevated levels of reactive oxygen species (ROS) can initiate oncogenesis by causing DNA mutations and genome instability (6). However, recent studies have shown that, once established, tumor cells are reliant on antioxidant pathways for growth and survival (7,8). Moreover, radio-and chemotherapies induce death or senescence of cancer cells partly by increasing ROS. Thus, acquiring a detailed understanding of the pathways and mechanisms that regulate stress response genes may le...

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Stress signals such as amino acid deprivation and redox imbalances activate the integrated stress response (ISR), which allows cells to alleviate the stress or to undergo apoptosis if the stress is unresolved. The ISR is also important for the survival of cancer cells, since they experience stress in the form of nutrient and oxygen deprivation and hence frequently activate stress-response pathways. Cellular stresses trigger up-regulation of the transcription factor ATF4, which subsequently activates stress response genes through recruitment to cis-regulatory sites known as C/EBP:ATF response elements (CAREs). Although the role of ATF4 in regulating stress response is well established, the identity of the C/EBP partner that heterodimerizes with ATF4 to execute this crucial function remains obscure. Here we show that the transcription factor C/EBPG is a critical partner of ATF4 and that C/EBPG:ATF4 heterodimers are the predominant CARE-binding species in stressed cells. Similar to ATF4, C/EBPG is necessary for resistance of MEFs to oxidative stress. MEFs lacking C/EBPG show increased levels of reactive oxygen species (ROS) as a consequence of impaired glutathione biosynthesis, as was also seen in ATF4-deficient MEFs. C/EBPG is required for stress-induced association of ATF4 with CAREs and the subsequent activation of several critical stress-responsive genes, including those with known pro-oncogenic functions. Accordingly, resistance to stress conferred by C/EBPG also facilitates the growth of cancer cells. Depletion of C/EBPG impairs the proliferation of cancer cells in vitro and elevates ROS levels. These effects can be suppressed by addition of the antioxidant, N-acetylcysteine. Mice lacking C/EBPG are smaller in size and show defective eye lens formation, similar to ATF4-deficient mice. The absence of C/EBPG also causes perinatal mortality due to pulmonary atelectasis and respiratory failure. This mortality can be rescued by in utero exposure to N-acetylcysteine. Accordingly, gene expression analysis suggests the presence of increased oxidative stress and impaired expression of stress-responsive genes in the lungs of Cebpg-/- newborn mice. Interestingly, Cebpg-/- mice on a mixed strain background, which do not show perinatal lethality, are resistant to the development of solid malignant tumors. These findings suggest that the role of C/EBPG as a stress response regulator may be important for tumor development/progression. A pro-oncogenic function for C/EBPG is also suggested by the observation that elevated C/EBPG levels are associated with poor patient prognosis in several clinical cancer studies. Activation of stress-responsive genes through upregulation of C/EBPG could be a mechanism deployed by cancer cells to mitigate the high levels of ROS and metabolic stresses that they experience. The importance of C/EBPG and its targets in tumor cells could potentially be exploited to devise novel anti-cancer therapies. Citation Format: Manasi K. Mayekar, Christopher J. Huggins, Nancy Martin, Karen L. Saylor, Mesfin Gonit, Parthav Jailwala, Manjula Kasoji, Diana C. Haines, Octavio A. Quinones, Peter F. Johnson. C/EBPG: A critical stress response regulator with a pro-oncogenic role. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR02.

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